Key Laboratory of Marine Drugs, Chinese Ministry of Education, Institute of Marine Drug and Food, Ocean University of China, Qingdao 266003, China.
Glycobiology. 2011 Jan;21(1):13-22. doi: 10.1093/glycob/cwq123. Epub 2010 Aug 20.
Porcine intestinal mucosa heparin was partially depolymerized by recombinant heparinase 1 (heparin lyase 1, originating from Flavobacterium heparinum and expressed in Escherichia coli) and then fractionated, leading to the isolation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel electrophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new compounds. The most prominent oligosaccharide comprises the common repeating unit of heparin, ΔUA2S--GlcNS6S-IdoA2S--GlcNS6S, where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-d-glucopyranose, IdoA is l-idopyranosyluronic acid, S is sulfate and n = 0-7. A second prominent heparin oligosaccharide motif corresponds to ΔUA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0-5 and GlcA is d-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in heparin. The prominence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the -GlcNS3S6S-IdoA2S- linkage is particularly susceptible to heparinase 1.
猪肠黏膜肝素经重组肝素酶 1（肝素裂解酶 1，源自Flavobacterium heparinum 并在大肠杆菌中表达）部分解聚，然后进行分级分离，得到 22 种均一的寡糖，大小从二糖到十六聚糖不等。这些寡糖的纯度通过凝胶电泳、强阴离子交换和反相离子对高效液相色谱法确定。使用电喷雾电离质谱法测定寡糖的分子量，并使用 600 MHz 的一维和二维核磁共振波谱法阐明其结构。所鉴定的寡糖中有 5 种代表新化合物。最突出的寡糖包含肝素的常见重复单元，ΔUA2S--GlcNS6S-IdoA2S--GlcNS6S，其中ΔUA 是 4-脱氧-α-l-赤式-己-4-烯吡喃糖醛酸，GlcN 是 2-脱氧-2-氨基-d-葡萄糖，IdoA 是 l-艾杜糖醛酸，S 是硫酸盐，n=0-7。第二个突出的肝素寡糖基序对应于 ΔUA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S（其中 n=0-5，GlcA 是 d-吡喃葡萄糖醛酸），这是肝素中抗凝血酶 III 结合位点的片段。这第二组寡糖的突出地位和完整的抗凝血酶 III 结合位点的缺失表明 -GlcNS3S6S-IdoA2S-键特别容易受到肝素酶 1 的作用。
